Transfusion medicine is an important part of modern health care and the provision of reliably phenotyped red blood cells (RBC) is essential for safe and effective blood transfusions. For identification of many RBC antigens, monoclonal antibodies of either murine or human origin are available for use in agglutination assays, in which they perform as well as or better than the human polyclonal antibody preparations which they have replaced. However, the detection of some blood groups is still reliant on the use of human polyclonal antisera, which is a less reliable reagent source with respect to availability, batch to batch variation and bio-safety. The use of recombinant antibody and phage display technology for the discovery of new monoclonal antibodies with specificity for some of these RBC antigens has the potential to deliver an economical, unlimited supply of specific antibody reagents suitable for use in RBC phenotyping. Samples of human B cells from donors producing useful phenotyping antibodies were identified and transformed using Epstein Barr virus into lymphocyte cell lines. Antibody genes were obtained from the cell lines in the form ofRNA which was reverse transcribed, amplified by PCR and cloned into a phagemid vector system to generate several combinatorial antibody libraries. These antibody libraries were displayed on the surface of phage particles and subjected to antigen-driven selection by several rounds of phage display biopanning using soluble and cell based RBC antigens. In addition a large naIve library was biopanned against the same antigens in an attempt to isolate a wide range of antibodies suitable for blood typing. Several high quality combinatorial antibody libraries with respect to size (> 107 clones) and diversity were generated. Biopanning of recombinant libraries resulted in enrichment of phage antibodies specific for RBC antigens, and several clones were isolated which were shown to be specific for Duffy a antigen. The isolated antibodies would be ideal candidates for re-engineering into multivalent antibody molecules capable of direct agglutination of RBC and as such, have the potential to replace human polyclonal sera in the identification of Duffy a RBC antigen phenotyping.
| Identifer | oai:union.ndltd.org:ADTP/258305 |
| Date | January 2007 |
| Creators | Fiddes, Jane L. Sutton, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. Biotechnology & Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Fiddes Jane L. Sutton., http://unsworks.unsw.edu.au/copyright |
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